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Am J Physiol Heart Circ Physiol 293: H1282-H1290, 2007. First published May 18, 2007; doi:10.1152/ajpheart.00910.2006
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Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts

Baiqiu Wang, Amer Omar, Tatjana Angelovska, Vanja Drobic, Sunil G. Rattan, Stephen C. Jones, and Ian M. C. Dixon

Institute of Cardiovascular Science, St. Boniface General Hospital Research Centre and Department of Physiology, University of Manitoba, Winnipeg, Canada

Submitted 23 August 2006 ; accepted in final form 17 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transforming growth factor-beta1 (TGF-beta1) signal and downstream Smads play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 (I-Smad7) is an inducible regulatory Smad protein that antagonizes TGF-beta1 signal mediated via direct abrogation of R-Smad phosphorylation. The effect of ectopic I-Smad7 on net collagen production was investigated using hydroxyproline assay. Adenovirus-mediated I-Smad7 gene (at 100 multiplicity of infection) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-beta1 in primary rat cardiac myofibroblasts. In I-Smad7-infected cells, we also observed the ablation of TGF-beta1-induced R-Smad2 phosphorylation vs. LacZ controls. Overdriven I-Smad7 was associated with significantly increased expression of immunoreactive 65-kDa matrix metalloproteinase-2 (MMP-2) protein in culture medium of myofibroblast compared with LacZ-infected cells. Expression of the 72-kDa MMP-2 variant, e.g., the inactive form, was not altered by exogenous I-Smad7 transfection/overexpression. Furthermore, I-Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced [3H]thymidine incorporation vs. Ad-LacZ-infected controls. We suggest that negative modulation of R-Smad phosphorylation by ectopic I-Smad7 may contribute to the downregulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of I-Smad7 may provide a new therapeutic strategy for cardiac fibrosis.

myofibroblast; cardiac remodeling; matrix metalloproteinase; p27; fibroblast proliferation; cardiac fibrosis

AFTER LEFT VENTRICULAR (LV) myocardial infarction (MI), the infarcted myocardium undergoes wound healing and accelerated matrix deposition (59), a critical component of cardiac wound healing. In time, interstitial fibrosis of the noninfarcted or remnant heart also occurs and is associated with increased myocardial stiffness. Excessive wound repair is characterized by marked interstitial cardiac fibrosis, and myofibroblast proliferation contributes to the development of cardiac hypertrophy and heart failure (23).

Transforming growth factor-beta (TGF-beta) is a major regulator of cardiac fibrosis during the development of cardiac hypertrophy (13, 29, 44). TGF-beta enhances matrix deposition by increasing the synthesis of matrix components such as collagen and fibronectin (30). Moreover, TGF-beta promotes the expression of inhibitors of extracellular matrix proteases, such as plasminogen activator inhibitor and tissue inhibitors of matrix metalloproteinases (TIMPs) while simultaneously downregulating interstitial collagenases that function to degrade matrix components (39, 45). We have previously demonstrated the activation of matrix metalloproteinase-2 (MMP-2) in murine heart after large MI (49).

TGF-beta signaling is initiated through binding to a transmembrane heteromeric type I (TbetaRI) and type II (TbetaRII) receptor complex that normally displays serine/threonine kinase activity (62). After the activation of the receptor, the signal is transferred to the nucleus through Smad proteins (16, 26). The Smad family consists of receptor-regulated Smads (R-Smad, Smad2, and Smad3), common mediator Smad (Co-Smad4), and inhibitory Smads (I-Smad6 and I-Smad7; see Ref. 31). Upon TGF-beta binding, R-Smad is phosphorylated by TbetaRI and complexes with Co-Smad 4, which translocates to the nucleus to interacting with DNA-binding partners and transcriptional coactivators and corepressors (11, 61). Cellular responsiveness to TGF-beta signaling is controlled via mechanisms in the extracellular space, at the membrane, and in the cytoplasm and nucleus.

A well-described function of inhibitory I-Smad7 lies in the autoinhibition of R-Smad signaling; to do this, it associates with activated TbetaRI to inhibit phosphorylation and activation of R-Smads (35). Although TGF-beta is a major regulator of I-Smad7 activation, other cytokines, i.e., interferon-{gamma} and tumor necrosis factor-{alpha}, are known to contribute to transcriptional regulation of this protein (34, 54). I-Smad7 antagonizes growth inhibition and the induction of apoptosis and embryonic lung morphogenesis induced by TGF-beta1 (20, 21, 43, 64). Because long-term (7 days at 10 ng/ml) TGF-beta1 treatment inhibits [3H]thymidine incorporation and proliferation of myofibroblasts in culture (37), we hypothesized that this is the result of reciprocal activation of I-Smad7. Thus we sought to characterize the effect of ectopic I-Smad7 in our in vitro cardiac myofibroblast system on p27 as a key point of inhibition of cell cycle progression. We also examined the proliferative effect of the ectopic overexpression of I-Smad7 in cardiac myofibroblast using the [3H]thymidine incorporation technique.

Our data reflect a relatively early decrease in I-Smad7 expression in infarct scar and remnant LV tissues from post-MI hearts, indicating a potential role of I-Smad7 in the development of cardiac fibrosis in post-MI heart (58) wherein cardiac myofibroblasts contribute to cardiac repair. Furthermore, bleomycin-induced lung fibrosis, which is mediated by consistent activation of TGF-beta1, is prevented by the administration of exogenous (adenoviral delivery) I-Smad7 (36). Nevertheless, surprising little is known of control of matrix turnover by I-Smad7 in cardiac myofibroblasts. Thus we sought to define I-Smad7 modulation of collagen in cultured cardiac myofibroblasts by addressing collagen synthesis and MMP expression in cells overexpressing I-Smad7.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary adult cardiac myofibroblast culture. Adult cardiac myofibroblast cultures were established from ventricular tissue of male Sprague-Dawley rats according to the methods of Brilla et al. (5) with minor modifications (22). Adult rats (175–200 g body mass) were killed, and hearts were subjected to Langendorff perfusion with a flow of 5 ml/min at 37°C with recirculating Joklik's medium containing 0.1% collagenase (chaotropic agent) and 2% BSA for 25–35 min. Liberated cells were collected by centrifugation at 2,000 rpm for 10 min. Cells were resuspended in DMEM-F-12 and plated on a 100-mm noncoated culture flask at 37°C with 5% CO2 for 2 h. Cardiac fibroblasts attached to the bottom of the culture flask during a 2-h incubation, whereas nonadherent myocytes were removed by changing the culture medium. The cells were maintained in DMEM-F-12 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10–4 M ascorbate. The cells used for the study were from the second passage (P2); TGF-beta1 treatment was initiated after 48 h incubation of cells in DMEM-F-12 in the absence of serum. I-Smad7 adenovirus was added to the cultured cells 24 h before TGF-beta1 stimulation. The purity of fibroblasts used in these experiments was found to be ≥95%, using routine phenotyping methods described previously (22). Briefly, endothelial cells were labeled with the use of a monoclonal antibody against factor VIII, and we found that less than ~1% of cultured cells were positive for this protein. Less than 1% of cells were positive for desmin, which is specific for smooth muscle cells, and >95% of cells stained positively for procollagen type 1, which is a major protein product of fibroblasts. However, passaged fibroblasts (P1 and P2 cells) that have been cultured in DMEM-F-12 containing 10% FBS for up to at least 5 days demonstrated significant markers for myofibroblast phenotype, i.e., {alpha}-smooth muscle actin and SMemb.

Recombinant adenovirus. Replication-deficient human adenovirus vector expression mouse I-Smad7 under the control of CMV promoter (AdvCMVSmad7) is kindly provided by Dr. Anita Roberts (NIH, Bethesda, MD). Expression of the ecotopic I-Smad7 in cardiac myofibroblasts was confirmed by Western blot with anti-flag antibody (Sigma, Oakville, Ontario, Canada). AdvCMVLacZ encoding beta-galactosidase was chosen as a control in the experiments. In situ X-Gal staining was used to confirm the infection efficiency of I-Smad7 adenovirus at 100 multiplicity of infection (MOI) is >95% in cardiac myofibroblasts.

Protein extraction and assay. Cardiac myofibroblasts infected with I-Smad7 adenovirus (100 MOI) in the absence or presence of TGF-beta1 (10 ng/ml) for indicated time were washed with cold PBS, and total proteins were isolated using RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (10 mM NaF, 1 mM sodium orthovanadate, and 20 mM beta-glycorophosphate). The cell lysate was incubated on ice for 1 h and then sonicated for 5 s (repeated 3 times). After centrifugation at 16,000 g for 15 min at 4°C, the supernatant was used for the total protein assay. Total protein concentration of all samples was measured using the bicinchoninic acid method as described previously (46).

Western blot analysis of target proteins. Prestained low-molecular-weight marker (Bio-Rad, Hercules, CA) and 20 µg of protein from samples were separated on 10 or 12% SDS gels by SDS-PAGE. Separated protein was transferred on a 0.45 µM polyvinylidene difluoride (PVDF) membrane that was blocked at room temperature for 1 h or overnight at 4°C in Tris-buffered saline with 0.2% Tween 20 (TBS-T) containing 5% skim milk and probed with primary antibodies for 1 h at room temperature. The primary antibody against phosphorylated R-Smad2 was diluted 1:250 in 0.2% TBS-T with 5% skim milk, whereas the total R-Smad2 primary antibody was diluted 1:200 in 0.2% TBS-T with 5% skim milk. The primary antibody for actin was diluted 1:500 in 0.2% TBS-T with 5% skim milk. Phosphorylated (P) extracellular signal-regulated kinase (ERK) and P-c-Jun NH2-terminal kinase (JNK) antibodies were diluted 1:250 in 0.2% TBS-T with 5% skim milk, and p27 primary antibodies were diluted 1:1,000; phospho-Rb primary antibodies were diluted 1:500. Secondary antibodies included horseradish peroxidase (HRP)-labeled anti-rabbit for phosphorylated R-Smad2 and anti-mouse for total R-Smad2, actin, P-ERK, and P-JNK. All secondary antibodies were diluted 1:10,000 with 0.2% TBS-T with 1% skim milk and incubated for 1 h at room temperature. Protein bands on Western blots were visualized by ECL Plus (Amersham, Arlington Heights, IL) according to the manufacturer's instructions and were developed on film or using a Molecular Dynamics chemiluminescence detector (Storm 860; Amersham-Pharmacia Baie d'Urfé, PC, Canada). Relatively even protein loading was confirmed by immunoblotting against actin.

Immunodetection of MMP-2, MT1-MMP, and TIMP2. Cardiac myofibroblasts were cultured until confluent, and the medium was changed to DMEM-F-12 in the absence of serum. After a 24-h incubation, I-Smad7 (100 MOI), LacZ (100 MOI) adenovirus, and TGF-beta1 (10 ng/ml) were added to the medium. The conditioned media was collected at 24 h TGF-beta1 stimulation and/or I-Smad7 adenovirus administration. The expression of MMP-2, MT1-MMP, and TIMP in the I-Smad7-infected cells was examined by Western blot analysis. Sample proteins (i.e., conditioned media) were prepared under reducing condition, and separated on 10% SDS-PAGE (for MMP-2 and MT1-MMP) or 12% SDS-PAGE (for TIMP-2). Separated proteins (40 µg) were electrically transferred on PVDF membranes (Bio-Rad). After the nonspecific background was blocked with 5% milk in TBS-T, PVDF membranes were incubated with the antibodies for anti-rabbit polyclonal MMP-2 (AB809; 1:1,000), MT1-MMP (AB8221; 1:1,000), and TIMP-2 (AB8107; 1:1,000) for 1 h at room temperature. After the membrane was washed with Tween 20 buffer, the membranes were incubated with goat anti-rabbit HRP (1:10,000) for 1 h at room temperature. The immunoreactive bands were then visualized by ECL Plus detection (Amersham) according to the manufacturer's instructions and were developed on film. The even loading was confirmed by incubating membranes in Ponceau S solution (0.1% Ponceau S in 5% acetic acid; Sigma).

Hydroxyproline assay. To test the total collagen production, primary cardiac myofibroblasts were seeded in equal numbers in 100-mm cell culture dishes and grown until confluent in DMEM-F-12 with 10% FBS and 50 µg/ml ascorbic acid. Cells were starved for 48 h before the addition of TGF-beta1 and I-Smad7 adenovirus. After 72 h treatment, cells were scraped on the cell culture medium of 100-mm dishes. Both the cell lysate and culture medium were removed and placed in test tubes, and net collagen production was determined with a hydroxyproline-based assay as described previously (56). The result was expressed as degree of increase against values from control cardiac myofibroblasts.

[3H]thymidine incorporation. Proliferation assays were done on cells cultured on 24-well plates at a density of 0.2 x 106 cells/well (counted with a hemocytometer) in DMEM-F-12 with 10% FBS. After the cells reached ~60% confluency, medium was replaced with DMEM-F-12 + 0% FBS, 10% FBS, or 2% FBS. Cells were infected with 100 MOI I-Smad7 adenovirus vector for 24 h. Other groups of cells were starved for 24 h and then 10 ng/ml of TGF-beta1 were added to the media for 24 h. Cells were then pulsed for 4 h at room temperature with 2.0 µCi/ml [methyl-3H]thymidine (Amersham Pharmacia Biotech). DNA in cell lysate was precipitated with cold 20% TCA and filtered through 24-mm GF/A filters (Fisher). Beta emission was measured with Cytoscint scintillation fluid (ICN Pharmaceuticals, Costa Mesa, CA) and a scintillation counter (LS6500; Beckman Coulter, Fullerton, CA).

Reagents. Primary antibodies against P-ERK, P-JNK, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibody specific to phosphorylated Ser465/467 R-Smad2 was obtained from Calbiochem (San Diego, CA). Total R-Smad2 primary antibody was purchased from Cell Signaling (Beverly, MA). The primary antibodies against MMP-2, MT1-MMP, and TIMP-2 were obtained from Chemicon (Temecula, CA). HRP-labeled anti-mouse and HRP-labeled anti-rabbit secondary antibodies were purchased from Bio-Rad. TGF-beta1 peptide was purchased from R&D Systems (Minneapolis, MN).

Statistics. All values are expressed as means ± SE. One-way ANOVA followed by Student-Newman-Keuls post hoc analysis was used to compare the differences among multiple groups (SigmaStat, Point Richmond, CA). Significant differences among groups were defined by P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Net collagen content in cardiac myofibroblasts overexpressing ectopic I-Smad7. To determine if the protein level of the main component of extracellluar matrix, i.e., collagen, is altered by overexpression of ectopic I-Smad7, total collagen content was determined in adult cardiac myofibroblasts using hydroxyproline assay. As shown in Fig. 1, data were expressed as degree of increase units against the mean control value for each experiment. Exposure of cardiac myofibroblasts to TGF-beta1 (5 ng/ml) for 3 days was associated with marked increase of total collagen production compared with the control group. However, infection of the adult cardiac myofibroblasts with I-Smad7 adenovirus resulted in significant attenuation of net collagen production in the absence and presence of TGF-beta1 treatment compared with LacZ-infected myofibroblasts.


Figure 1
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  		Fig. 1. Histographical representation of qualified data from hydroxyproline-based assay in adult cardiac myofibroblasts treated with transforming growth factor (TGF)-beta1, inhibitory (I)-Smad7 adenovirus, and LacZ control virus. P ≤ 0.05 vs. control (*), vs. LacZ (#), and vs. LacZ + TGF-beta1 (&). Data are expressed as means ± SE (n ≥ 3 experiments).

 
Prolonged activation of P-Smad2 by TGF-beta1 in adult primary cardiac myofibroblasts. Although it is known that TGF-beta1 functions to activate R-Smads in different cell types, we examined the specific activation pattern of phosphorylated Smad2 in primary cardiac myofibroblasts. The cells were cultured with TGF-beta1 at indicated time points (5 min, 30 min, 1 h, 6 h, and 12 h) following starving in DMEM-F-12 containing 0% serum for 48 h. Phosphorylated Smad2 (P-Smad2) and total Smad2 expression were measured by Western blot analysis (Fig. 2A). We found that the expression level of P-Smad2 in quiescent cardiac fibroblasts is fairly low, and it was observed to increase at 5 min TGF-beta1 stimulation. It peaked at 30 min TGF-beta1 treatment and remained activated until 12 h treatment of TGF-beta1. Dose-dependent Smad2 activation by TGF-beta1 was also investigated in the current study (Fig. 2B); however, we found no significant alteration of P-Smad2 expression in cardiac myofibroblasts stimulated with different doses of TGF-beta1 ranging from 0.5 to 10 ng/ml.


Figure 2
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  		Fig. 2. Representative Western blot analysis of phosphorylated (P) Smad 2 in adult primary cardiac myofibroblasts treated with TGF-beta1. A: P-Smad 2 (58 kDa) increases at 5 min TGF-beta1 stimulation and remained activated until 12 h TGF-beta1 treatment. B: dose-dependent effect of 1 h TGF-beta1 stimulation on activation of Smad 2. No significant change of P-Smad 2 was found in cells treated with different doses of TGF-beta1. Western blots of actin (43 kDa) indicate relatively even protein loading among different lanes.

 
Blockade of expression of P-Smad2 by ectopic I-Smad7 in cultured cardiac myofibroblasts. As an intracellular negative regulator of TGF-beta signal, I-Smad7 was reported to inhibit the phosphorylation of Smad2 in a variety of cell types. To determine the effect of ectopic I-Smad7 on the expression and activation of Smad2 in cultured cardiac myofibroblasts, protein was extracted from myofibroblasts following overexpression of I-Smad7 via an adenovirus system. X-Gal staining showed >95% cells were positive in the infection of 100 MOI (data not shown). Furthermore, high-level expression of the protein in cardiac myofibroblasts was also confirmed by Western blot analysis using anti-flag antibody (data not shown). After adding I-Smad7 adenovirus to cells 24 h before TGF-beta1 stimulation, we observed that ectopic overexpression of I-Smad7 was associated with blockade of the synthesis of TGF-beta-induced P-Smad2 compared with LacZ controls, and this inhibitory effect is TGF-beta1 stimulation time point dependent. As shown in Fig. 3, P-Smad2 was significantly elevated following TGF-beta1 stimulation at 1, 2, and 6 h. Although ectopic I-Smad7 decreased the expression of Smad2 in the absence of TGF-beta1 treatment, it failed to block the activation of P-Smad2 induced by TGF-beta1 at the earlier time points, i.e., 1 and 2 h stimulation (Fig. 3, A and B). However, at 6 h TGF-beta1 treatment, this TGF-beta1-mediated R-Smad2 activation process was markedly attenuated by forced expression of I-Smad7 in cardiac myofibroblasts. Moreover, the expression level of total Smad2 was not altered across untreated or I-Smad7- and LacZ-infected cultures. We found the attenuation of Smad2 activation by ectopic I-Smad7 was achieved in a dose-dependent manner (Fig. 4).


Figure 3
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  		Fig. 3. Representative Western blot analysis of P-Smad 2 expression in primary cardiac myofibroblasts infected with adenoviral I-Smad7 [S7, 100 multiplicity of infection (MOI)] in the presence and absence of TGF-beta1 treatment (T; 10 ng/ml). A: expression of P-Smad 2 in adenovirus-infected myofibroblasts treated with TGF-beta1 at 1 h. B: expression of P-Smad 2 in adenovirus-infected myofibroblasts treated with TGF-beta1 at 2 h. C: expression of P-Smad 2 in adenovirus-infected myofibroblasts treated with TGF-beta1 at 6 h. Constitutive expression P-Smad 2 is dramatically decreased in ectopic I-Smad7-infected cells compared with cells infected with control LacZ adenovirus. However, the TGF-beta1-induced expression of P-Smad 2 was attenuated in myofibroblasts stimulated with TGF-beta1 at 6 h.

 

Figure 4
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  		Fig. 4. Dose-dependent effect of I-Smad7 on the activation of receptor-regulated (R)-Smad 2 in cardiac primary myofibroblasts.

 
Effect of overdriven I-Smad7 on mitogen-activated protein kinases activation in cardiac myofibroblasts. It is well known that TGF-beta can activate mitogen-activated protein kinases (MAPK) signaling pathway, including JNK and ERK (2, 18). As important downstream effectors of the TGF-beta signal pathway, they have also been implicated in TGF-beta-regulated collagen deposition (15, 60). We suggest that the inhibitory role of I-Smad7 on collagen accumulation in cardiac myofibroblast may be partially mediated by MAPKs. As indicated in Fig. 5, we found markedly decreased expression of P-ERK in I-Smad7 adenovirus-infected cells compared with control LacZ-expressing cells. However, the activation of ERK induced by 1 h TGF-beta1 stimulation was not completely blocked by ectopic I-Smad7 protein. Additionally, forced expression of I-Smad7 exerted no effect on activation of JNK in cardiac myofibroblasts in the presence or absence of 1 h TGF-beta1 treatment.


Figure 5
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  		Fig. 5. Representative Western blot analysis of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation in cardiac myofibroblasts infected with adenoviral I-Smad7 (S7, 100 MOI) in the presence and absence of TGF-beta1 treatment (10 ng/ml).

 
Regulation of synthesis of MMP components and TIMPs by I-Smad7 in cultured cardiac myofibroblasts. TGF-beta has been implicated in the modulation of MMPs, a family of proteolytic enzymes that function to degrade all the matrix components of the heart and thus serve in cardiac matrix turnover. To assess whether ectopic I-Smad7 exerts effect on the cardiac expression of MMPs, we obtained conditioned cell medium from cardiac fibroblasts infected with Ad Smad7 at 100 MOI. Western blot was then carried out to examine the expression level of MMP-2 in the culture medium. The data revealed that overdriven I-Smad7 significantly increased expression of immunoreactive 65-kDa MMP-2 protein (the active form of this MMP) in culture medium of myofibroblasts compared with LacZ-infected cells (Fig. 6). Expression of the 72-kDa MMP-2 variant (i.e., inactive form of this MMP) was not altered by exogenous I-Smad7 transfection/overexpression. Given that the activation process of MMP-2 requires the formation of a ternary complex composed of pro-MMP-2, MT1-MMP, and TIMP-2, immunodetection of these proteins was performed on the same samples to further investigate the possibility that alterations in these protein expressions may lead to the accelerated processing of pro-MMP-2 into MMP-2. We observed a significant increase of pro-MT1-MMP in the culture medium of cardiac myofibroblasts infected with I-Smad7 adenovirus compared with LacZ-infected controls (Fig. 6). However, no difference regarding synthesis of TIMP-2 was defined in cells overexpressing I-Smad7.


Figure 6
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  		Fig. 6. Regulation of matrix metalloproteinases (MMPs) and tissue inhibitor of MMP (TIMP)-2 synthesis by I-Smad7. Adult cardiac myofibroblasts were starved for 24 h before the addition of TGF-beta1. Adenovirus was added 12 h before TGF-beta1 stimulation. Expression of active form MMP-2 as well as MT1-MMP was upregulated in cardiac myofibroblasts overexpressing adenoviral I-Smad7 compared with control LacZ.

 
Influence of ectopic I-Smad7 on expression of p27 and phospho-Rb. Overexpressed I-Smad7 was associated with a significant increase of the p27-to-beta-tubulin ratio in myofibroblasts in 2% FBS-DMEM vs. values derived from Lac-Z-infected controls, noninfected 10% serum-treated cells, and 2% noninfected serum-treated cells (Fig. 7). Conversely, ectopic I-Smad7 was associated with a significant decrease of the ratio phospho-Rb/beta-tubulin protein band intensity in myofibroblasts (in 2% FBS-DMEM media) vs. values of Lac-Z-infected cells in 2% serum as well as noninfected 2 and 10% serum cultured cells (Fig. 7). Thus I-Smad7 overexpression was associated with a decreased level of phospho-Rb protein under these current culture conditions.


Figure 7
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  		Fig. 7. Representative Western blot of p27 expression (A) and pRb expression (B). Histographic representation of band absorbance ratio in adult primary cultured cardiac myofibroblasts (P1) infected with 100 MOI I-Smad7 adenovirus for 24 h in the presence or absence of 2% FBS-DMEM-F-12 and LacZ control virus (in 2% FBS-DMEM-F-12). P ≤ 0.05 vs. 2% FBS-DMEM-F-12 control (*), vs. LacZ (#), and vs. 10% FBS-DMEM-F-12 ({gamma}). Data are expressed as means ± SE for a pool of 5 experiments. Data are quantified by densitometric scanning. C: p27/ beta-tubulin relative band absorbance. D: pRb/beta-tubulin relative band absorbance.

 
Ectopic I-Smad7 is associated with decreased cardiac myofibroblast proliferation. The effect of ectopic I-Smad7 on primary myofibroblast [3H]thymidine incorporation in low-serum conditions (FBS-DMEM-F-12) was tested. Thymidine incorporation was significantly inhibited by I-Smad7- vs. Lac-Z-infected controls in 2% FBS-DMEM-F-12 as well as noninfected 2% serum-treated and 10% serum-treated cells (Fig. 8).


Figure 8
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  		Fig. 8. A: histographic representation of [3H]thymidine incorporation expression in adult primary cultured cardiac myofibroblasts (P1) treated with 10 ng/ml TGF-beta1 for 24 h, in the presence or absence of 2% FBS-DMEM-F-12. *P ≤ 0.05 vs. 2% FBS-DMEM-F-12 control. Data are expressed as means ± SE, using a pool of 3 experiments under identical conditions. B: histographic representation of quantified data from [3H]thymidine incorporation in adult primary cultured cardiac myofibroblasts (P1) infected with 100 MOI I-Smad7 adenovirus for 24 h (in the presence or absence of 2% FBS-DMEM-F-12) and LacZ control virus (in 2% FBS-DMEM-F-12). P ≤ 0.05 vs. 2% FBS-DMEM-F-12 control (*), vs. LacZ (#), and vs. 10% FBS-DMEM-F-12 ({gamma}). Data are expressed as means ± SE of 4 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Excessive collagen deposition or pathological fibrosis is an important contributor to LV dysfunction. The abnormal deposition of collagen in various etiologies of heart disease has a dual effect. Although its tensile strength protects the injured heart from dilatation and rupture of the infarcted region, excessive matrix deposition leads to cardiac fibrosis, which impairs the already attenuated cardiac function. Therefore, antifibrotic agents that target steps in collagen synthesis and degradation pathways represent promising strategies for these diseases. TGF-beta is an important player in the fibrosis of a variety of tissues, including cardiac tissues. Previous evidence of blockade of TGF-beta signal with antisense, neutralizing antibody or decorin has highlighted the therapeutic potential by targeting this molecule in pathological fibrosis (1, 19, 27). Following the binding of the TGF-beta ligand to TbetaRII, TbetaRI is activated, which leads to the phosphorylation of R-Smads, including Smad2 and Smad3, and subsequently initiates the transduction of TGF-beta signal. On the other hand, activated TbetaRI receptor also functions to induce the expression of I-Smad7, which antagonizes TGF-beta signaling either by competing for binding of R-Smads to TbetaRI, thereby blocking R-Smads from interacting with the receptor (14, 52), or by increasing ubiquitin-mediated degradation of TGF-beta receptor (25). Although touted as a putative means to block canonical TGF-beta signaling (58), information of the effects of I-Smad7 expression is incomplete.

Some lines of evidence have pointed to the negative regulatory role of I-Smad7 in the activation of R-Smads in a variety of tissues. For example, overexpression of I-Smad7 was shown to attenuate Smad2/3-mediated inhibition of embryonic morphogenesis (63, 64). In bleomycin-induced lung fibrosis, ectopic I-Smad7 was also associated with inhibition of Smad2 phosphorylation, decreased type I procollagen mRNA, and net collagen production (36). Moreover, blockade of activation of Smad2 and downregulation of collagen type I, III, and IV was observed in renal tubular epithelial cells (50) and smooth muscle cells (24) overexpressing I-Smad7. In hepatic stellate cells, ectopic expression of I-Smad7 also leads to abrogation of Smad2 activation (8). Data from our laboratory have shown that I-Smad7 expression was decreased in both scar tissue and remnant tissue in post-MI rat hearts, accompanied by decreased cytosolic expression of phosphorylated Smad2 (58). Although this in vivo study implies that abnormal expression of I-Smad7 may play a role in cardiac fibrosis, the underlying mechanisms remain unclear.

In this study, we found that the marked prolonged activation of Smad2 by TGF-beta1 was effectively blocked by ectopic expression of I-Smad7 in both a dose-dependent and time-dependent manner, suggesting I-Smad7 prevented TGF-beta1-mediated phosphorylation of Smad2 in cardiac myofibroblasts. However, this inhibitory effect was specifically on the activation process, given the fact that adenovirus-mediated I-Smad7 overexpression did not alter total Smad2 expression. Moreover, consistent with our published in situ immunostaining analysis showing decreased expression of both collagen type I and type III in primary cardiac myofibroblasts overexpressing ectopic I-Smad7 (58), we now demonstrate that overdriven I-Smad7 significantly attenuated total collagen synthesis induced by TGF-beta1. Together the data indicate that blockade of Smad2 activation by overexpression of exogenous I-Smad7 may result in inhibition of TGF-beta1-induced collagen deposition. This association between Smad2 activation and synthesis of collagen also supports that phosphorylated Smad2 may play a major role in the transcriptional regulation of collagen promoter, a process requiring nuclear translocation of P-Smad2. To further investigate whether nuclear translocation of P-Smad2 was also involved in the regulation of collagen production by I-Smad7, we performed immunofluorescence analysis of Smad2 localization following I-Smad7 adenovirus administration. However, no significant inhibition of the nuclear translocation of P-Smad2 by overexpression of I-Smad7 was found in adult cardiac myofibroblasts (data not shown). Therefore, attenuation of collagen synthesis in cardiac myofibroblasts caused by overdriven I-Smad7 may be mediated by decreased expression of P-Smad2 and not by blockade of nuclear translocation of P-Smad2.

In addition to traditional Smad-mediated transcriptional activation, TGF-beta-induced activation of the MAPK pathways has also been shown to play a role in TGF-beta-responsive biological consequences, including collagen deposition (40, 55). Although cross talk between Smad proteins and MAPKs has been implicated in the modulation of Smad phosphorylation and translocation, MAPK pathway activation may precede and be independent from Smad protein activation (10). To address the possible effect of I-Smad7 on the activation of JNK and ERK kinases, we performed Western blot analysis of these MAPKs in cardiac myofibroblasts overexpressing I-Smad7 protein. Although we observed attenuation of P-ERK expression by overdriven I-Smad7, the 1-h-TGF-beta1-induced activation of ERK was not ablated by I-Smad7 protein, suggesting that blockade of ERK activation via I-Smad7 may be achieved in a TGF-beta-independent manner. In addition to the induction of JNK by TGF-beta1, ectopic expression of I-Smad7 has been indicated to synergistically cause a significant stimulation of JNK activity in Mv1Lu cells (32). However, in cardiac myofibroblasts overexpressing I-Smad7 proteins, we did not observe induction of JNK activity either by TGF-beta1 or overdriven I-Smad7 protein. Thus the data indicate that ERK may play a more important role than JNK in I-Smad7-mediated downregulation of collagen production in cardiac myofibroblasts.

MMPs play a crucial role in the remodeling process of myocardium following MI (47, 51). MMP-2, also known as gelatinase A, is important in maintenance of cardiac matrix, since this metalloproteinase may process denatured collagen and gelatins. The mechanism underlying activation of pro-MMP-2 is known to implicate other MMPs such as MMP-1 (7), autoactivation (3), and the urokinase plasminogen system (33). MMP-2 activation is also mediated by MT-MMP1 (41), which requires the assistance of TIMP-2, which functions as a linker between MT1-MMP and pro-MMP-2 to form a ternary complex at the cell surface (48). Few studies have revealed the importance of I-Smad7 in the activation process of MMPs, and we found that ectopic I-Smad7 protein was associated with accelerated activation of pro-MMP-2 in cardiac myofibroblasts. The concomitant increase of MT1-MMP expression is notable, since it may contribute to the proteolytic cleavage of pro-MMP-2 to MMP-2. Although TIMP-2 is also necessary for the proteolysis of MMP-2 (4), the role of TIMP-2 in cardiac myofibroblasts may be secondary, since its expression is unchanged in the presence of overdriven ectopic I-Smad7 when compared with controls. Nevertheless, because the binding of TIMP-2 to MMP-2 as well as MT1-MMP affects the enzymatic activity of MMP-2 (6), further study may be needed to clarify the precise role of TIMP-2 in the regulation of MMP-2 activation by I-Smad7.

MMP expression can be modified at the transcriptional level by a variety of physiological signals, including growth factors and cytokines. Despite the induction of MMP-2 by I-Smad7, exposure of the quiescent cardiac myofibroblasts to TGF-beta1 was not associated with any marked alteration of MMP-2 activity as observed in other cell types (9, 57). As mentioned above, I-Smad7 has been shown to be able to exert a TGF-beta-independent regulatory effect on gene transcription (38), and thus whether activation of MMP-2 by I-Smad7 acts via the TGF-beta signal remains unclear. Because we found that the treatment of myofibroblasts with TGF-beta led to upregulation of MT1-MMP, the effect of overexpression of I-Smad7 parallels that of the ligand. This could be caused either by complete disruption of the TGF-beta-I-Smad7 regulatory loop, or more likely, by a TGF-beta-independent I-Smad7 signaling path. The latter possibility seems plausible in view of the recent suggestion that I-Smad7 is complex, and possibly pleuripotent (12). We suggest that overdriven I-Smad7 may induce the disruption of MT1-MMP-dependent processing of pro-MMP2, partially contributing to downregulated collagen synthesis.

The application of agents that target myofibroblast cell cycle progression and thus proliferation represent promising strategies for managing global cardiac fibrosis. The cyclin-dependent kinase inhibitors are important in cell cycle progression in normal cells (42, 53). In cultured myofibroblasts, we found that ectopic I-Smad7 is associated with a significant increase in p27 expression. Because p27 inhibits the formation of the cdk2/cyclin E complex, it is a salient marker for cell cycle inhibition. We also found that the phospho-Rb expression was significantly decreased by I-Smad7 overexpression in myofibroblasts, thus potentially limiting cell cycle progression at the G1 phase as previously suggested (17, 28). In combination with data showing reduced proliferation in transfected cells, we suggest that altered p27 and phospho-Rb expression may be rate-limiting factors in regulating the cell cycle in myofibroblasts when influenced by ectopic I-Smad7.

In conclusion, we provide evidence that ectopic expression of I-Smad7 appears to inhibit TGF-beta-induced collagen production in primary cardiac myofibroblasts. This antifibrotic effect of I-Smad7 involved both TGF-beta-dependent activation of Smad2 and TGF-beta-independent activation of MMP-2. Finally, overdriven I-Smad7 is also associated with inhibition of proliferation of myofibroblasts. Modification of TGF-beta signaling by ectopic I-Smad7 may allow for the development of therapeutic strategies to attenuate post-MI cardiac fibrosis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by grants from the Heart and Stroke Foundation of Manitoba and the Canadian Institutes of Health Research (CIHR Group in Experimental Cardiology). M. C. Dixon holds the Myles Robinson Heart Fund Scholarship at the University of Manitoba; B. Wang and T. Angelovska were supported by traineeships of the Manitoba Health Research Council at the University of Manitoba. A. Omar and V. Drobic were supported by studentships from the St. Boniface General Hospital and Research Foundation. The current project was also funded with an operating grant from the Heart and Stroke Foundation of Manitoba. B. Wang is a recipient of a postdoctoral fellowship from the Manitoba Health Research Council.


   ACKNOWLEDGMENTS
 
We thank Dr. Darren H. Freed for technical expertise and advice in completion of this work.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. M. C. Dixon, Institute of Cardiovascular Science, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (e-mail: idixon{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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